Until now, structures of very few membrane-proteins have been determined at atomic resolution. We plan to study molecular organization of photosystem I and determine its structure at atomic resolution. Photosystem I, the light-dependent plastocyanin-ferredoxin oxidoreductase, is a heteromultimeric pigment-protein complex in the photo synthetic membranes of chloroplasts and cyanobacteria. It contains at least eleven proteins, 100 chlorophylls, beta-carotenes and a series of electron transfer centers. Photosystem I is a typical membrane-protein complex. Moreover, it is stable, abundant and easy to purify. Thus photosystem I is a suitable model system for structural analysis of membrane-protein complexes. We have embarked on a comprehensive project aimed to elucidate structure of photosystem I from the thermophilic cyanobacterium Mastigocladus laminosus. Our main objectives for this project are: (1) Topographical analysis of photosystem I organization. We will identify protein domains that are exposed on the surface of the complex or are involved in protein-protein interactions within the complex. NHS- biotinylation, protease accessibility, and chemical cross-linking studies will be used. The information generated by these methods will be used to orient structures of individual subunits in the complex. These data will also assist in correct identification of structural components revealed by X-ray crystallography. (2) Generation of simpler systems for structural analysis. We will determine primary structure of photosystem I proteins by cloning and sequencing the corresponding genes. We will overproduce peripheral subunits of photosystem I in E. coli and elucidate their structure by crystallography. We will also generate subunit-deficient mutants lacking the PsaF and PsaL proteins, thus engineering a complex that is simpler, more hydrophilic, but still functional. (3) X-ray diffraction analysis: We will crystallize entire and simpler photosystem l complexes as well as the overexpressed proteins. We plan to determine their structures from analysis of X-ray diffraction patterns. Finally we will integrate biochemical and X-ray crystallographic data to propose model for the detailed structure of photosystem I.